The stoichiometry of the cardiac sodium-calcium exchange system

A thermodynamic approach was adopted for determining the stoichiometry of the cardiac Na-Ca exchange system. Vesicles were equilibrated with 0.1 mM 45CaCl2 in a medium containing 30 mM NaCl, 20 mM KCl, and 110 mM LiCl. The vesicles were then treated with valinomycin and diluted into media containing the same 45CaCl2 and NaCl concentrations as in the equilibration medium but with the external KCl concentration adjusted so as to impose either positive or negative membrane potentials (delta psi). 45Ca2+ uptake ensued upon establishing a positive (inside) potential whereas 45Ca2+ efflux was observed for a negative delta psi. These delta psi-dependent Ca2+ movements did not occur if NaCl was omitted from the media, indicating that they were mediated by the Na-Ca exchanger. High concentrations of either NaCl or CaCl2 inhibited the delta psi-dependent Ca2+ movements. To determine the exchange stoichiometry, a series of Na+ gradients were established across the vesicle membrane so as to oppose the effects of a constant delta psi (either positive or negative) on Ca2+ movements. The stoichiometry n could be determined from the magnitude of the Na+ gradient that exactly compensated for delta psi such that no net Ca2+ movement occurred. This point is defined by the relation (n - 2)delta psi = nENa where ENa is the equilibrium potential for Na+ under these conditions. The value of n (+/- S.E.) determined in this way was 2.97 +/- 0.03 (n = 9).